Red blood cell surrogate

ABSTRACT

A red blood cell surrogate which is composed of a nanocrystalline core particle to which an oxygen carrier such as hemoglobin is bound. An oxygen carrier anchor coating is provided between the nanocrystalline core particle and the oxygen carrier in order to provide binding of the oxygen carrier to the nanocrystalline core particle without denaturing the oxygen carrier or otherwise destroying its oxygen carrying capacity. The nanocrystalline core particle with the oxygen carrier bound thereto is coated with a layer of lipid and then an outer layer of a sugar or allosteric effector is applied.

This is a continuation-in-part of application Ser. No. 08/029,773 whichwas filed on Mar. 3, 1993, now U.S. Pat. No. 5,306,508, which is acontinuation-in-part of application Ser. No. 08/000,199 which was filedon Jan. 4, 1993, now U.S. Pat. No. 5,334,394, which is acontinuation-in-part of application Ser. No. 07/690,601, now U.S. Pat.No. 5,178,882, which is a continuation-in-part of application Ser. No.07/542,255, which was filed on Jun. 22, 1990, now U.S. Pat. No.5,219,577.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to red blood cells and thedevelopment of compounds which can be used to replace or supplement redblood cells. More particularly, the present invention relates tosynthetic compounds which have the same oxygen transport capabilities asred blood cells and can be used in vivo and in vitro as a red blood cellsurrogate.

2. Description of Related Art

There has been, and continued to be, a great deal of interest indeveloping suitable blood substitutes. One avenue of investigationinvolves the use of free hemoglobin solutions. However, free monomericand polymeric as well as cross-linked hemoglobin solutions exhibitosmolarities which are much greater than normal blood. The highosmolarity tends to produce complications of vascular volume andperfusion pressures. Another problem is that unmodified hemoglobin isfreely filtered at the glomerulus and is nephrotoxic.

The use of free hemoglobin in a blood substitute is further complicatedby contaminants which may be present in the hemoglobin source or whichmay be introduced during the manufacturing process. Endotoxins andphospholipids are typical examples of contaminants which may beproblematic in deploying hemoglobin solutions as blood substitutes. Inaddition, heterogenous sources of hemoglobin have been shown to producemarked antibody titers in rats. Also, immunogenic complications havealso been reported in human trials.

Another approach to blood substitutes involves the use ofperfluorocarbons. These blood substitutes typically contain from 10-20%perfluorocarbon by weight and contain particle sizes of approximately150 nm. The perfluorocarbon type blood substitutes have been reported tohave adequate oxygen delivery capacity. However, perfluorocarbonsexhibit a linear oxygen dissociation curve which is not desirable foroptimum oxygen transport. In addition, perfluorocarbons have severalclinical complications. Perfluorocarbons are easily trapped in bloodfilter organs, resulting in depatomergaly and splenomegaly over a periodof some weeks to months. Further, perfluorocarbons exhibit cytotoxiceffects and are readily adsorbed by erythrocytes. This adsorptionresults in decreased cellular flexibility which could impair passage ofthe erythrocytes through capillary beds.

The encapsulation of hemoglobin in liposomes is another type of bloodsubstitute which has been investigated. Liposome encapsulation ofhemoglobin was first successfully used as a blood substitute about 40years ago. At that time, hemoglobin was encapsulated in a collodionmembrane. Liposome encapsulation has been successful in lowering thetoxicity of biological agents and increasing their circulation time.Cholesterol, phosphatidylcholine and other lipids have been used toproduce encapsulated hemoglobin capsules having diameters on the orderof 200 to 500 microns.

Liposome encapsulated hemoglobin appears to overcome many of thecomplications presented by the use of perfluorocarbons and freehemoglobin. However, some clinical complications still exist. Forexample, problems with reticuloendothelial uptake, platelet aggregationand suppression of the reticuloendothelial system have been experienced.As is apparent, there presently is a continuing need to provide newsynthetic compositions which can function effectively as a substitutefor red blood cells. The synthetic composition must be capable ofproviding acceptable levels of oxygen transport without causingundesirable side effects. The synthetic material should be easily storedand have a reasonable shelf-life. The blood substitute should beamenable to use in a wide variety of situations including resuscitationin field trauma following massive blood loss or blood replacement duringsurgery. In addition, the blood substitute should be amenable to invitro use to perfuse organs such as hearts, livers and kidneys duringtransport and transplantation.

SUMMARY OF THE INVENTION

In accordance with the present invention a red blood cell surrogate isprovided which can be used as a blood substitute in a wide variety ofsituations. The red blood cell surrogate has a nanocrystalline corewhich is composed of a ceramic metal or polymer material. The surface ofthe particle is coated with an oxygen carrier anchor material. An oxygencarrier is bound to the anchor coating and an exterior layer of lipid isprovided to complete the red blood cell surrogate.

As a feature of the present invention, the oxygen carrier anchormaterial substantially covers the surface of the nanocrystallineparticle to form a glassy film which has a threshold surface energy thatis sufficient to anchor the oxygen carrier to the core particle withoutdenaturing the oxygen carrier which is bound thereto. This allows theuse of a wide variety of core particle materials regardless of theirrespective surface energies. The anchor materials are basic sugars andallosteric effectors.

As another feature of the present invention, hemoglobin is a preferredoxygen carrier. It was discovered that hemoglobin can be anchored to theglassy film surrounding the nanocrystalline core without beingdenatured. The anchored hemoglobin provides oxygen transport in the samemanner as the hemoglobin found in naturally occurring red blood cells.

As a further feature of the present invention, the exterior surface ofthe red blood cell surrogate is coated with layer of lipid provides ared cell surrogate which more closely mimics the structure andfunctioning of naturally occurring red blood cells. The lipid layer isdesigned to mimic the outer membrane of red blood cells.

As an additional feature of the present invention, an outer layer ofsugar or allosteric effector may be provided as a cover for the lipidlayer. This outer layer helps to prevent coagulation activation and theresultant formation of intravascular thrombosis.

The red cell surrogates of the present invention are well-suited for usein any situation where there is a need for delivery of oxygen to tissuein vivo or in vitro. The red blood cell surrogates may be used in vivofor resuscitation in field trauma following massive blood loss or as ablood replacement for blood loss in surgery. In addition, the red bloodcell surrogate may be used in patients who are incapable of producingred blood cells owing to some disease process. In vitro use of the redblood cell surrogate includes perfusion of organs such as hearts, liversand kidneys during transport and storage prior to transplantation. Inaddition, the red blood cell surrogates may be used in cell culturesystems as an optional oxygen delivery system.

The above discussed and many other features and attendant advantages ofthe present invention will become better understood by reference to thefollowing detailed description.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The red blood cell surrogates in accordance with the present inventionare based on nanocrystalline core particles (diameters of less than1,000 nm) which are coated with an oxygen carrier anchor coating. Anoxygen carrier, such as hemoglobin, is bound to the oxygen carrieranchor coating. The nanocrystalline core particle with the oxygencarrier bound thereto, is then coated with an exterior layer of lipid. Afinal exterior coating of a sugar is then applied to cover the lipidlayer. The red blood cell surrogates mimic the oxygen carryingcapability of naturally occurring red blood cells and have been founduseful in transporting oxygen in the same manner as red blood cells bothin vitro and in vivo.

The red blood cell surrogates of the present invention are similar insome respects to the viral decoy vaccines disclosed in U.S. Pat. No.5,178,882 which issued on Jan. 12, 1993, and is owned by the sameassignee as the present application. U.S. Pat. No. 5,178,882 disclosesdecoy viruses made up of nanocrystalline core particles on which variousviral fragments or protein coatings are attached. The core particles arecoated with a surface modifying agent to promote anchoring of the viralparticles without denaturization thereof. The present invention is basedupon this same principal except that hemoglobin or other oxygen carrieris bound to the surface energy modifying layer (i.e., oxygen carrieranchor coating) and an exterior coating of lipid is provided. Thecontents of U.S. Pat. No. 5,178,882 are hereby incorporated byreference.

The nanocrystalline particles which form the core of the red blood cellsurrogate are made from a wide variety of inorganic materials includingmetals, ionic solids or ceramics. The core material may also bepolymers. The particles may range in size up to about 1000 nm. Thepreferred particle sizes are on the order of about 10 to 200 nm.Preferred metals include chromium, rubidium, iron, zinc, selenium,nickel, gold, silver, and platinum. Preferred ceramic materials includesilicon dioxide, silicon nitride, titanium dioxide, aluminum oxide,ruthenium oxide and tin oxide. The core particles may be made frommaterials such as diamond, calcium hydroxide, calcium oxide, calciumacetylsalicylate, calcium ascorbate, calcium carbonate, calcium citrate,calcium cyclamate, calcium gluconate, calcium glycerophosphate, calciumhypophosphate, calcium iodate, calcium lactate, calcium oxalate,phosphate, calcium pyrophosphate, calcium D-saccharate, calciumstearate, calcium succinate, calcium sulfite, calcium tartrate andcalcium phosphate compositions. Preferred polymers include polystyrene,nylon and nitrocellulose. Particles made from tin oxide, titaniumdioxide, calcium oxide, calcium hydroxide, or calcium phosphate diamondare particularly preferred.

Particles made from the above materials having diameters less than 1000nanometers are available commercially or they may be produced fromprogressive nucleation in solution (colloid reaction), or variousphysical and chemical vapor deposition processes, such as sputterdeposition (Hayashi, C. J. Vac. Sci. Technol. A5(4), Jul./Aug. 1987,pgs. 1375-1384; Hayashi, C., Physics Today, Dec. 1987, pgs. 44-60; MRSBulletin, Jan 1990, pgs. 16-47). Tin oxide having a dispersed (in H₂ O)aggregate article size about 140 nanometers is available commerciallyfrom Vacuum Metallurgical Co. (Japan). Other commercially availableparticles having the desired composition and size range are availablefrom Advanced Refractory Technologies, Inc. (Buffalo, N.Y.).

Plasma-assisted chemical vapor deposition (PACVD) is one of a number oftechniques that may be used to prepare suitable microparticles. PACVDfunctions in relatively high atmospheric pressures (on the order of onetorr and greater) and is useful in generating particles having diametersof up to 1000 nanometers. For example, aluminum nitride particles havingdiameters of less than 1000 nanometers can be synthesized by PACVD usingAl(CH₃)₃ and NH₃ as reactants. The PACVD system typically includes ahorizontally mounted quartz tube with associated pumping and gas feedsystems. A susceptor is located at the center of the quartz tube andheated using a 60 Khz radio frequency source. The synthesized aluminumnitride particles are collected on the walls of the quartz tube.Nitrogen gas is used as the carrier of the Al(CH₃)₃. The ratio ofAl(CH₃)₃ : NH₃ in the reaction chamber is controlled by varying the flowrates of the N₂ Al(CH₃)₃ and NH₃ gas into the chamber. A constantpressure in the reaction chamber of 10 torr is generally maintained toprovide deposition and formation of the ultrafine nanocrystallinealuminum nitride particles. PACVD may be used to prepare a variety ofother suitable nanocrystalline particles.

Nanocrystalline Calcium hydroxide may be prepared by streaming 50 mlvolumes of 0.75M of Calcium chloride and 1.5N Sodium hydroxide againsteach other over 10 ml of 100 mM sodium citrate during 400 W sonicationin a cup horn for 15 minutes [4° C.]. The resultant white opaque solidis washed 2 times with HPLC grade sterile water and sonicated at 450 W[4° C.] for an additional hour and adjusted to 150 mg/ml to provide adispersion of nanocrystalline particles which may be coated as describedbelow.

In order to attach hemoglobin or other oxygen carrier to the coreparticle, it must first be coated with a substance that provides athreshold surface energy to the particle which is sufficient to bind thehemoglobin without denaturing it. The core particles are preferablycoated by suspending them in a solution containing the dispersed surfacemodifying agent. It is necessary that the coating make the surface ofthe particle more amenable for attachment of hemoglobin or other oxygencarrier. Suitable coating substances in accordance with the presentinvention are basic sugars including cellobiose, trehalose, isomaltose,nystose sorbitol, lactitol, maltose and sucrose. In addition, allostericeffectors such as pyridoxal-5-phosphate; 2,3-phosphoglycerate, andsodium citrate may be used. If desired, the surface modifying coating(i.e., oxygen carrier anchor coating) can be made from a combination ofbasic sugars and allosteric effectors. It is only necessary that thecoating provide the surface of the nanocrystalline particle with athreshold energy which is sufficient to bind hemoglobin or other oxygencarrier without denaturing the relevant oxygen transport sites.

Sugars which exhibit the molecular stabilization required in accordancewith the present invention are polyhydroxylic in nature and have arelatively low molecular weight which affords them maximum rotationfreedom in the glassy state. Accordingly, it is expected that lowmolecular weight molecules including disaccharides and related alcoholsrich in hydroxy groups will afford the energetics that are classicallyassociated with water and yet be of sufficiently small molecular weightto produce functional glasses which provide the molecular stabilizationrequired in accordance with the present invention.

The coating solution into which the core particles are suspendedcontains, for example, from 1 to 30 weight/volume percent of the coatingmaterial. The solute is preferably double distilled water (ddH₂ O). Theamount of core particles suspended within the coating solution will varydepending upon the type of particle and its size. Typically, solutionswith particle densities of about 100 mg/ml or greater are suitable forsynthesis of the red blood cell surrogates.

The core particles are maintained in dispersion in the coating solutionfor a sufficient time to provide uniform coating of the particles.Sonication is the preferred method for maintaining the dispersion.Dispersion times ranging from 30 minutes to a few hours at roomtemperature are usually sufficient to provide a suitable coating to theparticles. The thickness of the coating is preferably less than 5nanometers. Thicknesses of the coating may vary provided that the finalcore particles include a uniform oxygen carrier anchor coating oversubstantially all of the particle surface.

The previously described calcium hydroxide nanocrystalline particles maybe coated with sodium citrate as follows: 50 ml of 100 mM sodium citrateis added to 50 ml of the freshly sonicated calcium hydroxidenanocrystalline particle dispersion (150 mg/ml) and lyophilizedovernight under low heat. The next morning the solid is rehydrated to 50ml, sonicated at 450 W for 5 minutes, and washed three times with HPLCgrade water. The preparation adjusted to 300 mg/ml for furtherhemoglobin carrier synthesis as described below.

Another exemplary coating procedure for calcium hydroxidenanocrystalline particles is as follows: 10 ml of 500 mM cellobiose isadded to 50 ml of the freshly sonicated calcium hydroxidenanocrystalline particle dispersion (150 mg/ml) and lyophilizedovernight under low heat. The next morning the solid is rehydrated to 50ml, sonicated at 450 W for 5 minutes, and washed three times with HPLCgrade water. The preparation is adjusted to 300 mg/ml for furtherhemoglobin carrier synthesis as described below.

A further exemplary coating procedure for calcium hydroxidenanocrystalline particles is as follows: 10 ml of 100 mg/mlpyridoxal-5-phosphate is added to 50 ml of the freshly sonicated calciumhydroxide nanocrystalline particle dispersion (150 mg/ml) andlyophilized overnight under low heat. The next morning the solid isrehydrated to 50 ml, sonicated at 450 W for 5 minutes, and washed threetimes with HPLC grade water. The preparation is adjusted to 300 mg/mlfor further hemoglobin carrier synthesis as described below.

The oxygen carrier which is attached to the coated nanocrystalline coreparticle is preferably hemoglobin. The hemoglobin may be derived fromnumerous sources including human, bovine, or ovine. Purified hemoglobinis available commercially from many different sources. In addition, anyof the well known techniques for isolating and purifying hemoglobin fromblood may be used. Hemoglobin is the preferred oxygen carrier; however,other oxygen carrying macromolecules may be utilized if desired.Hemoglobin may be produce through recombinant engineering techniques ortransgenic animals such as swine.

The hemoglobin may be attached to the coated nanocrystalline coreparticles by a wide variety of procedures. Preferably, a solution ofpurified hemoglobin is added to a solution of coated nanocrystallinecore particles and agitated for a sufficient time to allow binding ofthe hemoglobin to the coated particles. Although it is preferred thatthe binding of hemoglobin to the coated core particles occur insolution, many other techniques may be used provided that intimatecontact between the purified hemoglobin and the coated core particle isprovided.

The amount of hemoglobin which is bound to the coated nanocrystallineparticle may vary widely depending upon the intended use for the redblood cell surrogate. It is preferred that the amount of hemoglobinbound to each nanocrystalline particle be maximized as much as possiblewithout adversely affecting the oxygen transport properties of thesurrogate. When maximum oxygen transport is desired, then maximumbinding of hemoglobin to the nanocrystalline particle is desired.However, when lesser degrees of hemoglobin bound to the coatednanocrystalline particles may be reduced. Preferably, an excess ofhemoglobin is combined with the coated nanocrystalline particles toassure maximum hemoglobin binding.

The next step in forming the red blood surrogate of the presentinvention involves coating the nanocrystalline particles with a lipid.The lipids used to coat the nanocrystalline particle and boundhemoglobin are the same lipids commonly used to form liposomes. Suitablelipids include phospholipids such as phosphatidylcholine, cholesteroland phosphatidylserine. The lipid layer is applied to thenanocrystalline core particle and bound hemoglobin in the same manner asthe anchor coating and hemoglobin.

It is not clear whether the core particle and bound hemoglobin need tobe totally covered with a lipid layer. Preferably, the amount of lipidused to coat the core particles and bound hemoglobin will be an excessto ensure complete interaction and coating of the particles.

The final step in forming the red blood cell surrogates of the presentinvention involves coating the lipid layer with an outer coating. Theouter coating may be made from the same sugars and allosteric effectorswhich are used to form the anchor coating. The outer coating ispreferably applied by lyophilization of the particles in the presence ofthe sugar or allosteric effector. Cellobiose is a preferred outercoating. The outer coating may also be applied using any of theconventional coating processes. Preferably, the outer coating will coversubstantially the entire surface of the red blood cell surrogate. Thisouter coating preferably will have a thickness on the order of a fewnanometers. This outer stabilizing layer is designed to preventactivation of blood coagulation mechanisms in vivo and thereby reducethe possibility of intravascular thrombosis.

The red blood cell surrogates in accordance with present invention maybe stored in a variety of forms. Preferably, the red blood cellsurrogates are freeze dried and stored in a dry form. However, ifdesired, the red cell surrogate may be stored in the form of a solution.The red blood cell surrogates may be used alone or in combination withany number of other blood substitutes. When in a freeze-dried form, thered blood cell surrogates may be reconstituted with any of the wellknown aqueous pharmaceutical carriers. These pharmaceutical carriersinclude buffered saline, with or without allosteric effectors, plasmaand whole blood. Solutions composed of phosphate buffered saline (PBS)and albumin are preferred carriers.

The red blood cell surrogates may be injected in vivo as concentrated ordilute suspension. Use of the red blood cell surrogate in vitro willalso range from direct addition of a freeze-dried surrogate to theaddition of concentrated or dilute surrogate suspensions. In general,the red blood cell surrogates of the present may be used in the samemanner as naturally occurring red blood cells. Further, the red bloodcell surrogates may also be used in the same manner as other syntheticblood substitutes. Examples of practice are as follows:

EXAMPLE 1

The following example demonstrates the preparation of a red blood cellsurrogate in accordance with the present invention. The fabricationprocess involved coating ultrafine nanocrystalline diamond particleswith a glassy film of disaccharides and then physically adsorbingpurified hemoglobin. The assembly was then encapsulated within simpleliposomes.

One (1.00) g. of acid cleaned commercial ultrafine synthetic diamondparticles (GE Series 300, Worthington, Ohio) was dispersed in 5.0 ml of100 mM cellobiose (Sigma) solution with 175 watt sonication (Branson)for 10 minutes. The colloid was then incubated at 4.0° C. overnight in a10 kD stir cell. The following day, this colloid was lyophilized for 24hours and reconstituted in 1.0 ml of ddH₂ O. Unabsorbed cellobiose wasremoved by 10 kD stir cell ultra filtration (UF) (Filtron) against 100ml of 20 mM phosphate buffer (pH 7.4) (PRB) and corrected to 2.0 ml.(UF) (Filtron) against 100 ml of 20 mM phosphate buffer (pH 7.4) (PRB)and corrected to 2.0 ml.

Five hundred (500) mg of human hemoglobin type A_(o) (Sigma) wasdissolved in 5.0 ml PBS (pH 6.8) (Gibco) and then ultrafiltered against150 ml PRB at 4.0 C. in a 50 kD stir with 30 psi N2. The filtrate wasadjusted to 3.0 ml. The surface modified diamond dispersion (2.0 mL) wasthen added to the 50 kD ultrafiltrate cell and allowed to incubateovernight with slow stirring (5 psi N₂). The filtrate was adjusted to3.0 ml. The surface modified diamond dispersion (2.0 mL) was then addedto the 50 kD ultrafiltrate cell and allowed to incubate overnight withslow stirring (5 psi N2, 4.0° C.). The next morning, 35 uL ofphosphatidyl dipalmitoyl serine (10 mM of 6.0 mM NaOH) (Sigma), 50 uL ofphosphatidyl dipalmitoyl choline [6.8 mM stock] Sigma, and 8.7 uL ofcholesterol [3.9 mM stock] (Sigma) was stirred in and incubated forapproximately 6.0 hours. The final product was again filtered in a 50 kDultrafiltration cell over 30 psi N₂ against 20 ml PBS (pH 7.4, 4.0° C.and adjusted to 5.0 ml for an estimated hemoglobin concentration of 10g/dL.

EXAMPLE 2

In this example, red blood cell surrogates were made in the same manneras Example 1 except that pyridoxal-5-phosphate is substituted forcellobiose as the oxygen carrier anchor coating.

50 mg of acid cleaned commercial ultrafine synthetic diamond particles(GE Series 30, Worthington, Ohio) was dispersed by 175 watt sonication(Branson) for 10 minutes, mixed with 75.0 mg of pyridoxal-5-phosphate[Sigma] and adjusted to 10.0 ml with deionized water. The mixture wasspun lyophilized overnight, washed with 4-10 ml aliquots of deionizedwater and reconstituted to 25 mg/ml in pH 7.40, 20 mM phosphate buffer.

4.0 ml [25 mg/ml] of nanocrystalline core particles were added to 1.0 mlrecovered red blood lysate hemoglobin [26.10 g/dL]. The mixture was thenslowly dialyzed into 100 ml of 0.5X PRB overnight at 4.0 C. under anitrogen head of 20 psi and through a 10 kD ultrafiltration cell. Thenext morning, 34 uL of phosphatidyl serine [10 mM of 6.0 mM NaOH](Sigma), 50 uL of cholesterol [3.9 mM stock] (Sigma) was stirred in anincubated for approximately 6.0 hours. The final product was againfiltered to remove free hemoglobin in a 50 kD ultrafiltration cell over30 psi N₂ against 200 ml PBS (pH 7.4, 4.0° C.) and adjusted to 1.0-2.5ml or an estimated hemoglobin concentration of 10 g/dL.

The preceding procedure was repeated at different pyridoxal-5-phosphateconcentrations. As a result, red blood cell surrogates were preparedwherein the nanocrystalline particles were coated in solutionscontaining 1 mM pyridoxal-5-phosphate and 30 mM pyridoxal-5-phosphate.

The red blood cell surrogates prepared in Examples 1 and 2 were analyzedfor oxygen dissociation characteristics as well as size distribution,electrophoretic mobility and retained molecular conformation. The redblood cell surrogates coated with cellobiose (Example 1) exhibited anoxygen dissociation (P50) of 26-30 mm Hg. The red blood cell surrogateshaving coatings of pyroxidal-5-phosphate should have oxygendissociations (P50) 37 mm Hg. This compares well with the oxygendissociation of whole human blood which is 31 mm Hg. In addition,hemoglobin-bound nanocrystalline particles were prepared in the samemanner as Example 2 except that the coating of lipid was deleted. Theoxygen-dissociation of the lipid-free hemoglobin-bound nanocrystallineparticles was 12 mm Hg.

The electrophoretic mobility of the red blood cell surrogates producedin Examples 1 and 2 were measured with Doppler electrophoretic lightscatter analysis (DELSA 440, Coulter Electronics, Inc., Hialeah, Fla.).The electrophoretic mobility was -1.7 um cm/Vs at a pH of 7.4 PRGBBUFFER at 25° C.

The size distribution of the red blood cell surrogates produced inExamples 1 and 2 were measured by both photon correlation spectroscopyat a 90° angle in PRB buffer at 22.5° C. (N4MD, Coulter) and bytransmission microscopy (TEM, Zeiss 190). The red blood cell surrogatesmeasured 187 nanometers plus or minus 37 nanometers by photoncorrelation. For electron microscopy, a 10 microliter drop of particlesin solution was placed on a paraffin surface which included acarbon-stabilized FORMVAR GRID (Ted Pella, Inc., Redding, Calif.) whichwas floated on top of the drop. Due to the high surface charge of theTEM GRID, the red blood cell surrogates absorbed to the grid allowingexcess solution to be removed by careful blotting. A similar method wasthen used to stain the particles with 2 % phosphotungstic acid. Thestained grid was then dried and the red blood cell surrogates identifiedas having particle sizes in the range of 50-100 nanometers.

The conformational integrity of the red blood cell surrogates wasverified by immunogold antibody affinity intensity. After beingdeposited on one nanometer TEM copper grids, the protein bound particleswere incubated for one hour at 27° C. with polyclonal rabbit anti-humanhemoglobin antibodies (Dako) and secondary goat anti-rabbit 30 nanometergold-labeled antibodies (Zymed Laboratories, San Francisco, Calif.). Thegold-labeled anti-bodies were observed to attach avidly to thehemoglobin present in the red blood cell surrogates.

EXAMPLE 3

In the following example, a red cell surrogate was prepared having abrushite core, cellobiose oxygen carrier anchor coating and hemoglobinoxygen carrier. The lipid layer was made from L-alpha-phosphatidylcholine, L-alpha-phosphatidyl serine and cholesterol. The outer coatingwas cellobiose.

The procedure used to prepare the red cell surrogate was as follows:

Preparing Calcium Phosphate Dihydrate (Brushite) NanocrystallineParticles:

Reagents. 0.75M CaCl₂ : 55.13 g CaCl₂.2H₂ O is dissolved with HPLC gradewater to 0.500 L in a volumetric flask. Filter sterilize with 0.2 umsterile filtration unit and place in a sterile 500 ml culture mediumflask. Store at room temperature.

0.25M Na₂ HPO₄ : 17.75 g of anhydrous Na₂ HPO₄ is dissolved with HPLCgrade water to 0.500 L in a volumetric flask. Filter sterilize with 0.2um sterile filtration unit and place in a sterile 500 ml culture mediumflask. Also store at room temperature.

Brushite synthesis. About a half hour before synthesis, prepare thesonicator by cooling down the cup horn. This is accomplished byadjusting the low temperature thermostat on the water condenser to 4° C.and dialing a setting of "4" on the peristatic circulator. Once the 4°C. mark is reached, prepare 50.0 ml of 0.75M CaCl₂ and 50.0 ml of 0.25MNa₂ H₂ PO₄ and load into 50 ml syringes. The syringes are then to beconnected to a 3-way luer lock connector so that they are set indiametric opposition--allowing the remaining luer port to be free todispel product. Once the mixing apparatus is set up, place a sterile 120ml sonicating flask in the cup horn and slowly power up the sonicator to100% power. Position the mixing apparatus so that the free luer port isover the sonicating flask. Expel syringe contents into the flask asrapidly and evenly as possible so as to empty each syringe roughly atthe same time. Then quickly secure a polypropylene liner over thesonicating flask and let sonicate for an additional 15 minutes.

Brushite washing. Roughly divide the prep into two 50 ml blue toppolypropylene tubes and pellet at 2000 rpm for 10 minutes (roomtemperature). Reconstitute by vortexing each pellet with sterile HPLCgrade water to 50 ml (or tube capacity) and pellet at 2000 rpm for 10minutes. Repeat this wash 3 more times and reconstitute the last pelletsto 50.0 ml. Transfer the dispersion to a sterile 120 ml sonicating flaskwith polypropylene liner. Place the flask in a previously cooledsonicator cup horn at 1° C. Sonicate at 100% power for 60 minutes.

Coating Brushite Nanoparticles With a Cellobiose:

Incubation/Lyophilization.

1. Sonicate the brushite (aqueous dispersion) prepared as described

above for 30 minutes at 25° C. at full power [175 Watts].

2. Then as quickly as possible, exchange suspending medium from water(stock) to a solution of 500 mM cellobiose using either a bench topmicrocentrifuge (30 seconds, full speed of 14,000 RPM) for small volumesor for larger volumes a floor models centrifuge (model 21K, in 50 mlcentrifuge tubes, 8,000 RPM for a maximum of 2 minutes). Suspend thepelleted carbon with 500 mM cellobiose, sonicate to aid dispersion(approximately 5 minutes at 25° C. at full power [175 Watts]) andfinally set the mixture on a rocking plate overnight in a cold room [4°C.].

3. The next day portion out the mixture into appropriately sized vesselsfor overnight lyophilization.

4. Leave the tubes capped with a layer of parafilm around the cap andplace them in a freezer until the washing step.

5. Reconstitute the brushite/cellobiose in a suitable buffer dependingon the application. Suitable buffers are low ionic strength bufferedphosphate (PRB), water, or bicarbonate. Reconstitution in the buffer isaccomplished by vortexing and a 5 minute sonication [175 Watts/25° C.].

6. Wash by repeated centrifugation (using either a bench topmicrocentrifuge [30 seconds, full speed of 14,000 RPM] for small volumesor for large volumes a floor model centrifuge [model 21K, in 50 mlcentrifuge tubes, 8,000 RPM for a maximum of 2 minutes]) andresuspension into the buffer.

7. Take a concentration measurement by removing 1 ml of the suspensiondehydrating it in a lyophilizer in a pre-weighed 1.7 ml Eppendorf tube,and massing.

8. Calculate the final volume necessary to bring the concentration to 1mg/ml. Add enough buffer to bring the concentration of thebrushite/cellobiose preparation to 1 mg/ml.

Final Preparation of Red Cell Surrogate:

The cellobiose coated brushite was then coated with hemoglobin asdescribed in Example 1. The coating of lipid and outer coating ofcellobiose were applied as follows:

All reagents were obtained from Sigma Biochemical (St. Louis, Mo.),unless otherwise noted. For a 100 ml brushite hemoglobin carriersynthesis, 6.60 g of egg yolk L-alpha-phosphatidyl choline, 0.660 g ofbovine brain L-alpha-phosphatidyl serine, and 3.10 g of cholesterol wasmixed in 132 mls of chloroform. Chloroform was removed by lyophilizationusing a Savant (Westbury, N.Y.) speed vac for at least 18 hours underlow heat in 50 ml Corex tubes. Following immediately afterlyophilization, 17 mls of a 500 mM solution of cellobiose was then addedto the phospholipids and corrected to 100 mls with sterile water. Themixture was then sonicated at 4.0° C. in a AES2000 Branson (Westbury,Conn.) sonifier for one hour at approximately 400 Watts and added to asterile 500 ml water jacketed reaction flask. The contents were thenlyophilized overnight under analogous conditions.

The red cell surrogates produced as described above were tested in thesame manner as Examples 1 and 2 and found to be effective as red bloodcell surrogates.

EXAMPLE 4

Red cell surrogates are made in the same manner as Example 3 except thatpyrodoxal-5-phosphate (P-5-P) is substituted for cellobiose as theoxygen carrier anchor coating. The coating of P-5-P is applied asfollows:

Pellet 100 ml of the dispersion prepared in Example 3 so that the entirecontents can be transferred to a 50 ml conical tube. Adjust the tubevolume to 40.0 ml. Then transfer the contents in 10 ml aliquots to four15 ml conical tubes. Dissolve 1000 mg of Pyroxidal-5-phosphate with 800μl of 10N NaOH and adjust with water to 10 mls. Filter sterilize thisclear yellow solution with a 0.2 μm acrodisc and add 2.5 ml aliquots toeach of the previously prepared 4 brushite tubes. Vortex each tube a fewseconds to make certain that the contents are well dispersed. Lyophilizeovernight [approx. 16 hrs] at the low drying rate setting. The nextmorning resuspend in 50 ml aliquots of sterile HPLC grade water fivemore times. Pellet once more and transfer the pellets to four 15 mlconical tubes and adjust the final preparation volume with water to 40.0ml.

As another option, P-5-P may also be substituted for cellobiose as theouter coating.

EXAMPLE 5

Red cell surrogates are made in the same manner as Example 3 except thatcitrate is substituted for cellobiose as the oxygen carrier anchorcoating. The coating of citrate is applied as follows:

Brushite/Citrate. Pellet the 100 ml of the dispersion prepared inExample 3 so that entire contents can be transferred to a 50 ml conicaltube. Adjust the tube volume to 40.0 ml. Then transfer the contents in10 ml aliquots to four 15 ml conical tubes. Add 10 ml of 100 mM citrateto each of the 15 ml conicals and nutate for 30 minutes at roomtemperature. Lyophilize overnight [approx. 16 hrs] at the low dryingrate setting. The next morning resuspend in 50 ml aliquots of sterileHPLC grade water five more times. Pellet once more and transfer thepellets to four 15 ml conical tubes and adjust the final preparationvolume with water to 40.0 ml.

The above examples demonstrate that the red blood cell surrogates inaccordance with the present invention are effective oxygen transportparticles which may be used as a substitute for red blood cells both invitro and in vivo.

Having thus described exemplary embodiments of the present invention, itshould be noted by those skilled in the art that the within disclosuresare exemplary only and that various other alternatives, adaptations andmodifications may be made within the scope of the present invention.Accordingly, the present invention is not limited to the specificembodiments as illustrated herein, but is only limited by the followingclaims.

What is claimed is:
 1. A red cell surrogate comprising:a nanocrystallinecore particle, said core particle having a surface; an oxygen carrieranchor coating located on the surface of said nanocrystalline coreparticle; an oxygen carrier bound to said oxygen carrier anchor coating;a layer of lipid surrounding said oxygen carrier; and an outer layer. 2.A red cell surrogate according to claim 1 wherein said nanocrystallineparticle comprises a ceramic, metal or polymer.
 3. A red cell surrogateaccording to claim 1 wherein said oxygen carrier anchor coatingcomprises a glassy film which substantially covers the surface of saidnanocrystalline core particle, said glassy film having a thresholdsurface energy that is sufficient to anchor said oxygen carrier to saidcore particle without denaturing said oxygen carrier.
 4. A red cellsurrogate according to claim 3 wherein said glassy film comprises asugar selected from the group of basic sugars consisting of cellobiose,trehalose, isomaltose, nystose sorbitol, lactitol, maltose and sucrose.5. A red cell surrogate according to claim 3 wherein said glassy filmcomprises an allosteric effector selected from the group consisting ofpyridoxal-5-phosphate, 2,3-phosphoglycerate, and sodium citrate.
 6. Ared cell surrogate according to claim 3 wherein said glassy filmcomprises a basic sugar and an allosteric modifier.
 7. A red cellsurrogate according to claim 1 wherein said oxygen carrier compriseshemoglobin.
 8. A red cell surrogate according to claim 7 wherein saidoxygen carrier comprises human hemoglobin.
 9. A red cell surrogateaccording to claim 1 wherein said exterior layer of lipid comprises aphospholipid.
 10. A red cell surrogate according to claim 9 wherein saidphospholipid is selected from the group consisting ofphosphatidylcholine, cholesterol and phosphatidylserine.
 11. A red cellsurrogate according to claim 1 wherein said outer layer comprises asugar or allosteric effector.
 12. A red cell surrogate according toclaim 11 wherein said outer layer sugar is selected from the groupconsisting of cellobiose, trehalose, isomaltose, nystose sorbitol,lactitol, maltose and sucrose.
 13. A red cell surrogate according toclaim 11 wherein said outer layer allosteric effector is selected fromthe group consisting of pyridoxal-5-phosphate, 2,3-phosphoglycerate andsodium citrate.
 14. A composition of matter comprising:A. a red cellsurrogate comprising:a nanocrystalline core particle, said core particlehaving a surface; an oxygen carrier anchor located on the surface ofsaid nanocrystalline core particle; an oxygen carrier bound to saidoxygen carrier anchor coating; a layer of lipid surrounding said oxygencarrier; an outer layer; and B. a physiological acceptable carrier forsaid red cell surrogate.
 15. A method for delivering oxygen to cells,said method comprising the step of treating said cells with a red bloodcell surrogate, said surrogate comprising:a nanocrystalline coreparticle, said core particle having a surface; an oxygen carrier anchorcoating located on the surface of said nanocrystalline core particle; anoxygen carrier bound to said oxygen carrier anchor coating; a layer oflipid surrounding said oxygen carrier; an outer layer.
 16. A method fordelivering oxygen to cells according to claim 15 wherein saidnanocrystalline particle comprises a ceramic, metal or polymer.
 17. Amethod for delivering oxygen to cells according to claim 15 wherein saidoxygen carrier anchor coating comprises a glassy film whichsubstantially covers the surface of said nanocrystalline core particle,said glassy film having a threshold surface energy that is sufficient toanchor said oxygen carrier to said core particle without denaturing saidoxygen carrier.
 18. A method for delivering oxygen to cells according toclaim 17 wherein said glassy film comprises a sugar selected from thegroup of basic sugars consisting of cellobiose, trehalose, isomaltose,nystose sorbitol, lactitol, maltose and sucrose.
 19. A method fordelivering oxygen to cells according to claim 17 wherein said glassyfilm comprises an allosteric effector selected from the group consistingof pyridoxal-5-phosphate, 2,3-phosphoglycerate and sodium citrate.
 20. Amethod for delivering oxygen to cells according to claim 17 wherein saidglassy film comprises a basic sugar and an allosteric modifier.
 21. Amethod for delivering oxygen to cells according to claim 15 wherein saidoxygen carrier comprises human hemoglobin.
 22. A method for deliveringoxygen to cells according to claim 15 wherein said exterior layer oflipid comprises a phospholipid.
 23. A method for delivering oxygen tocells according to claim 15 said exterior layer of lipid comprises aphospholipid.
 24. A method for delivering oxygen to cells according toclaim 23 wherein said phospholipid is selected from the group consistingof phosphatidyl-choline, cholesterol and phosphatidylserine.
 25. Amethod for delivering oxygen to cells according to claim 15 wherein saidcells are located in vivo.
 26. A method for delivering oxygen to cellsaccording to claim 15 wherein said cells are located in vitro.
 27. Amethod for delivering oxygen to cells according to claim 15 wherein saidouter layer comprises a sugar or allosteric effector.
 28. A method fordelivering oxygen to cells according to claim 15 wherein said outerlayer sugar is selected from the group consisting of cellobiose,trehalose, isomaltose, nystose sorbitol, lactitol, maltose and sucrose.29. A method for delivering oxygen to cells according to claim 15wherein said outer layer allosteric effector is selected from the groupconsisting of pyridoxal-5-phosphate, 2,3-phosphoglycerate and sodiumcitrate.